The aim of the proposed work is to elucidate the role of myosin in the mechanism of muscle contraction by determining the rotational trajectory of the cross-bridge during energy transduction (ATP hydrolysis) and the generation of force. Several site-specific spectroscopic probes will be localized on spatially separated sites on the myosin cross-bridge. The probes will sense both the large scale movement of the peptide backbone or global cross-bridge rotational movement related to force production and local cross-bridge conformation change related to energy transduction. Steady-state and time-resolved spectroscopic techniques follow the orientation sensitive probe signals in various physiological states and after mechanical perturbations to provide the global orientation and dynamics of myosin. The time-resolved techniques, polarized fluorescence photobleaching recovery (PFPR) and time-resolved fluorescence polarization (TRFP), follow probe movement in muscle fibers on the submillisecond time domain. PFPR imposes an optical transient to probe order that relaxes to equilibrium without any perturbation of the muscle fiber system. TRFP follows probe rotational movement in response to a mechanical perturbation of the muscle fiber. These techniques, used on different physiological states of the fiber, provide independent but complementary information on cross-bridge order and dynamics. New methodology for combining data from multiple probe signals distinguishes local from global cross-bridge rotation. Different spectroscopic signals from the same probes interpreted in terms of probe-protein contacts provide the local conformation of the cross-bridge. A coupled dipole oscillator model of the probe-protein contacts interprets absorption, fluorescence, and circular dichroism signals from the probes in terms of the molecular structure of the complex. The combination of the local and global conformation of the cross-bridge will produce a comprehensive map of cross-bridge movement during the active cycle permitting the correlation of local energy transduction events with global force generating events.
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